Induction of Autotetraploids in Pummelo (Citrus grandis L. Osbeck) through Colchicine Treatment of Meristematically Active Seeds In Vitro

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1 Proc. Fla. State Hort. Soc. 123: Student Best Presentation Award 1st place Induction of Autotetraploids in Pummelo (Citrus grandis L. Osbeck) through Colchicine Treatment of Meristematically Active Seeds In Vitro Divya Kainth and Jude W. Grosser* University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL Aditional index words. Citrus paradisi, flow cytometry, interploidy crosses Autotetraploids in pink/red-fleshed pummelo selections , C2-5-12, and UKP-1 (all derived from Hirado Buntan pink pummelo) were produced by treating the germinated seeds with different colchicine concentrations of control, 0.1%, 0.2%, and 0.3% for different treatment durations of 12 and 24 h. The seedlings emerged when put on rooting media under sterile conditions. The seedling ploidy was determined via flow cytometry at a stage when the seedlings had 1 2 expanded leaves. Higher colchicine concentrations and treatment durations decreased the survival rates of the seedlings compared with the lower concentrations and smaller treatment durations. The seeds that received higher concentrations and longer durations turned brown completely or had a dead meristematic bud. A similar trend was observed for the number of mutated shoots (tetraploids and mixoploids). Colchicine treatment decreased the growth rate of the affected seedlings. The frequency of autotetraploidy varied among the selections. Selections , C2-5-12, and UKP-1 successfully produced 2, 1, and 3 autotetraploids and 1, 2, and 4 mixoploids, respectively. The most efficient colchicine concentration was 0.1%. Reversion of the tetraploids and mixoploids into diploids was also observed. The stable pink/red-fleshed tetraploid plants generated should be useful as breeding parents in grapefruit/ pummelo improvement programs. Use of monoembryonic tetraploids in interploid citrus crosses eliminates the need for embryo rescue to recover seedless triploid progeny; until now, no pink or red-fleshed monoembryonic pummelo parents are available. Acknowledgments. The authors thank FCPRAC (Florida Citrus Production Research Advisory Committee), NVDMC (New Varieties Development & Management Corporation), and Barney and Harriet Greene for financial support of this project. This research is part of the MS thesis research project of Divya Kainth. *Corresponding author; phone: (863) , ext. 1372; jgrosser@ufl.edu Grapefruit (Citrus paradisi Macf.), which originated in Barbados and is cultivated worldwide, is among the most popular fresh citrus species. The United States followed by China and South Africa are the biggest producers. Its growing demand and popularity exerts pressure on grapefruit breeders to come up with improved cultivars with traits to meet consumers expectations, such as dark red flesh color and seedlessness. Excessive number of seeds in citrus makes it unappealing to the consumers and unacceptable for local and international markets. Breeders worldwide are increasing efforts to come up with quality seedless cultivars. Seedlessness has been successfully achieved in the past in many cultivars through ploidy manipulations as triploidy is associated with sterility. Several approaches from traditional hybridization to molecular biotechnology have been applied in order to induce seedlessness in Citrus fruits. These include selection of spontaneous triploids from a natural population (Wakana et al., 1981) or those from 2x 2x crosses (Esen and Soost, 1971; Geraci, 1978; Geraci et al., 1975), in vitro somaclonal variation (Deng et al., 1985), endosperm culture (Chen et al., 1991; Gmitter et al., 1990; Wang and Chang, 1978), somatic hybridization between a diploid and a haploid (Kobayashi et al., 1997), genetic transformation (Koltunow et al., 1998), and interploidy hybridization between a diploid and a tetraploid parent (Esen and Soost, 1972). Out of these, the interploidal hybridization has been the most effective and is commonly used to produce seedless triploids. However, scarcity of tetraploids in the Citrus gene pool has given rise to the need to induce tetraploidy in breeding lines that could be used as parental material for the interploid crosses. Crosses where the tetraploid is used as the female parent to produce triploids is more efficient as a result of normal sexual fertilization between a diploid female gamete and a haploid male gamete (18n + 9) (Cameron and Burnett, 1978; Esen and Soost, 1972; Soost and Cameron, 1975). In such crosses, when tetraploid polyembryonic cultivars are used as a female parent, there arises a need to rescue the embryo via tissue culture techniques. This is to avoid the suppression and subsequent abortion of the zygotic hybrid embryo by the more vigorous nucellar seedlings. Though the identification and selection of the zygotic seedling is possible through morphological, isozyme, histochemical, cytological, and molecular techniques, it becomes a costly, labor-intensive, and time-consuming process. Use of a tetraploid monoembryonic female parent instead eliminates the need for embryo rescue, making the process much simpler and efficient. Considering the present market demands, scarcity of superior tetraploid parents in grapefruit and factors slowing the triploid breeding process, pummelos (Citrus grandis L. Osbeck) are beginning to be included as germplasm sources in grapefruit breeding programs. Pummelo, being the ancestor of grapefruit and because it is monoembryonic, has a comparatively greater diversity in its gene pool that makes it an ideal candidate to contribute towards grapefruit improvement. It is monoembyonic, hence each seedling is a unique genotype. Some of the pummelo types are lower in naringin and furanocoumarin content, which could be useful 44 Proc. Fla. State Hort. Soc. 123: 2010.

2 in reducing the levels of these chemicals in subsequent triploid hybrids. Use of such red-fleshed tetraploid pummelo types as a female parent in the interploidal crosses could produce hybrids that are more appealing and consumer friendly. Colchicine, an alkaloid obtained from meadow saffron (Colchicum autumnale), is a mitotic inhibitor (Blakeslee and Avery, 1937) and is commonly used to induce tetraploidy in breeding lines in Citrus. Barrett (1978) attempted to produce autotetraploids in monoembryonic cultivars by treating the axillary buds with colchicine in vivo. However, he had difficulty producing stable tetraploid plants. The most probable explanation for this was that he used bigger plants and it is difficult to double the chromosomes in large and broadly differentiated meristems (Sanford, 1983). Stable autotetraploids in monoembryonic cultivars have been produced by cochicine treatment of the axillary buds (Oiyama and Okudi, 1986), shoot tips (Juarez et al., 2004), and somatic embryogenic callus (Wu and Mooney, 2002). Lehrer et al. (2008) induced tetraploidy by treating germinated seeds of Japanese barberry (Berberis thunbergii var. atropurpurea) with colchicine and oryzalin in vivo. This technique has also been used to produce tetraploids in ornamental plants Syringa spp. L. (lilac) (Fiala, 1988), Rhododendron spp. L. (rhododendron) (Leach, 1961), and Iris spp. L. (iris) (McEwen, 1990). However, there is no report of tetraploid induction from seed treatment in Citrus. Also, there has not been any report on tetraploid induction in pummelo cultivars to date. This study reports an efficient method for induction of tetraploids by treatment of germinating seeds from elite pummelo selections with colchicine in vitro. It compares the effect of different colchicine concentrations and exposure durations of seedling growth and development; and tetraploid induction. Materials and Methods Plant materials. Seeds were extracted in from the fruits of red-fleshed pummelo selections , C2-5-12, and UKP-1 located at the Citrus Research and Education Center (CREC). The extracted seeds were washed under running water 3 4 times. The seeds were dried, peeled, and sterilized using 10% sodium hypochlorite for 8 min followed by 2 3 rinses with deionized water. Seeds were then placed onto seed germination medium under sterile conditions. The seeds were put on this medium for d until they germinated and the hypocotyl had emerged about 5 8 mm out of the cotyledons. At this point, the seeds were undergoing high meristematic activity and were ready to be treated. Colchicine treatments. The experimental design was a two-way factorial consisting of three colchicine concentrations and two exposure periods. There were three replications of each treatment. Colchicine stock solution was prepared by dissolving colchicine in a few drops of dimethylsulfoxide (DMSO) followed by the addition of sterile water to bring the final concentration to 1 g/ml. This solution was filter sterilized. Per treatment, 15 pregerminated seeds were placed in conical Falcon tubes containing 10 ml of liquid seed germination media with final colchicine concentrations of 1, 2, and 3 g/l. Seeds immersed in liquid seed germination media without colchicine were used as controls. The seeds exposed to different colchicine concentrations were exposed for periods of 12 or 24 h each. Flasks were put on the rotary shaker at 30 rpm for the respective exposure periods to facilitate the contact and penetration of colchicine in the meristems which were covered by the cotyledons. The rotary shaker was contained in a dark chamber maintained at 25 ± 2 C. After each treatment s respective exposure time, the seeds were taken out and placed on solid seed germination media and placed under dark conditions to facilitate seedling elongation. The seedlings were transferred to rooting media supplemented with naphthalene acetic acid (NAA) after about 2 weeks and were placed under 16-h light/8-h dark conditions for further growth. Emergent seedlings were analyzed for their ploidy via flow cytometry at a stage when the seedling had at least three fully expanded leaves. The seedlings confirmed to be tetraploid were micrografted onto vigorous rootstocks. The micrografted tetraploids were put under shade for d prior to moving them to the greenhouse with set points of 21 to 17 C day/night temperatures. Ploidy analysis. Ploidy was analyzed using a tabletop flow cytometer (Partec GmbH, Münster, Germany). This technique makes it possible to analyze genotypes per day. Flow cytometry works by estimating the volume and florescence of isolated nuclei. The ploidy is presented in form of a histogram of integral fluorescence with the peaks depicting the ploidy level of the respective sample. The protocol is a series of steps starting with excision of a 0.2- to 0.3-cm 2 piece of fully expanded leaf tissue and placed in a 50-mm plastic petri dish. The sample was prepared for analysis using a High Resolution Staining Kit (Partec GmbH). The tissue is chopped with a sharp razor blade after adding few drops of Nuclei Extraction Buffer. After chopping, 6 7 more drops of Nuclei Extraction Buffer were added and the sample was filtered through a yellow 50-µm filter into a 3.5-mL (55 mm 12 mm) Sarstedt tube. The staining buffer (DAPI) was added drop by drop through the filter to infiltrate the remaining cells, until half of the tube was filled. Each sample was incubated for s at room temperature before running it on the flow cytometer. The sample moves as a very narrow, laminar flowing sample stream through the flow cuvette. When the cells or the particles labeled with fluorescent coloring due to the staining buffer pass through the measuring area one after the other, the individual cells or particles get illuminated by the excitation light and the fluorescent light intensity which is proportional to DNA content is measured and analyzed to depict the respective number of chromosomes and hence the ploidy level of the sample. Results and Discussion The colchicine treatment induced tetraploidy in all the three pummelo selections. However, the frequency of tetraploids varied among treatments. Some treatments also produced cytogenic chimeric plants having tetraploid and diploid nuclei in varying proportions of cells. Chimeric plants have been recovered in similar in vitro studies conducted by Wu and Mooney (2002). Data was assessed by calculating the survival rate of the seedlings and tetraploid induction efficiency (TIE) for each treatment. Tetraploid induction efficiency was computed by the formula given below by Bouvier et al. (1994): TIE = % seedling survival % tetraploid seedlings The most important factors that determine the tetraploid induction efficiency are colchicine concentration and the exposure period for which seeds were exposed to colchicine. Higher colchicine concentrations and longer duration period hampers seedling growth, causes hyperploidy, browning, necrosis in the meristematic tissue and death of the seedling (Sanford, 1983). In this study, all colchicine treatments greatly decreased the growth Proc. Fla. State Hort. Soc. 123:

3 Table 1. Average height of recovered pummelo seedlings 5 weeks after treatments with three colchicine concentrations and exposure periods. Treatment UKP-1 C Control 12.4 ± 2.6 A 13.5 ± 2.21 A 12.8 ± 2.67 A 0.1% 12 h 1.2 ± 0.63 B 1.6 ± 0.5 B 1.5 ± 0.4 B 0.1% 24 h 1.2 ± 0.56 B 1.3 ± 0.51 B 1 ± 0.95 B 0.2% 12 h 0.9 ± 0.5 B 1.4 ± 0.35 B 1.2 ± 0.38 B 0.2% 24 h 1.1 ± 0.56 B 1.3 ± 0.6 B 1.1 ± 0.4 B 0.3% 12 h 0.5 ± 0.1 B 1.8 ± 0.64 B X 0.3% 24 h 1.1 ± 0 B 13.5 ± 2.21 B 1.6 ± 0 B Mean separation (in columns) by Duncan s multiple range test, 5% level. A Table 2. Survival rate and tetraploid induction efficiency (TIE) from treatments of selections , UKP1, and C of pummelo (Citrus grandis) with colchicine at three concentrations and two exposure periods. Seedling survival Mixoploid Tetraploid Treatment (%) no. no. TIE Control % 12 h % 24 h % 12 h % 24 h % 12 h % 24 h UKP-1 Control % 12 h % 24 h % 12 h % 24 h % 12 h % 24 h C Control % 12 h % 24 h % 12 h % 24 h % 12 h % 24 h rate of the treated seedlings in all three selections. Five weeks after the treatment, the control counterparts grew up to height ranging from 5 to 16 cm (2 to 6.3 inches) long whereas all the surviving treated seedlings remained stunted with a height 2.5 cm (1 inch). Average heights of the control seedlings at 28 d after colchicine treatments were 12.4, 13.5, and 12.8 cm (4.9, 5.3, and 5 inches) for selections , UKP1, and C2-5-12, respectively (Table 1). High mortality was observed in all the treatments. Overall seedling survival across all colchicine treatments was highest for selection , which had 30% seedling survival rate, followed by C2-5-12, having 25.6%, and UKP1 with 21.1% (Table 2). The control treatments without any colchicine exposure had 93% to 100% seedling survival rates. Higher mortality rate in the treated seedlings was due to the toxicity of colchicine. This explains B Fig. 1. Flow cytometry histograms representing pummelo seedlings from selection UKP-1 with (A) tetraploid profile, and (B) mixoploid profile. decreasing seedling survival when colchicine concentrations were increased or when seeds were given longer exposures to the chemical. At the lowest concentration of colchicine (0.1%), the surviving seedling percentage was around 50%. The survival rate dropped to 0 to 20% at 0.3% colchicine concentration. The seedling survival rate dropped to almost half when the exposure period was increased from 12 to 24 h for every concentration. These results indicate that the seedling survival rate is inversely proportional to the concentration and exposure period of colchicine. Figure 1 shows an example of histograms obtained from the ploidy analyzer for a non-chimeric tetraploid and chimeric samples. The most effective concentration at which the tetraploids were regenerated was 0.1%, though too few tetraploids were recovered to draw strong conclusions. This in agreement with Oiyama and Okudai (1968), who previously reported that 0.1% of colchicine was the best concentration for tetraploid induction in shoot tips in citrus. Concentration of 0.2% at 12 h of exposure period also was able to produce two stable tetraploids, one in each selection and C2-5-12, respectively. Table 2 lists number of tetraploids and mixoploids obtained from each treatment as well as the corresponding tetraploid induction efficiencies. 46 Proc. Fla. State Hort. Soc. 123: 2010.

4 A B C Fig. 2. (A) Browning of a pummelo seedling epicotyl before the leaves emerged; (B) necrosis of a pummelo seedling and subsequent death after the emergence of the leaves; and (C) a stable tetraploid confirmed by flow cytometry, micrografted onto a vigorous rootstock. There were a total of five tetraploids regenerated from the three selections. Different selections generated varying numbers of tetraploids and mixoploids. Three tetraploids were produced in selection UKP1 two in and one in C There were also three mixoploids obtained from UKP1, one from and two from C Mixoploids are commonly found when the targeted tissue is muticellular. In such cases, a few cells are mutagenized and the others remain diploid. When these partially mutated meristems differentiate to form plant organs, a mixture of tetraploid and diploid tissue is observed. Tetraploid induction efficiency is a good measure to find out the most effective treatment as it takes into account both seedling survival rate and number of tetraploids produced. The highest TIE of 1.0% was obtained from treatment with 0.1% concentration of colchicine and 12-h exposure period in selection UKP1. Treatments with higher colchicines concentrations all with 0.3%, most with 0.2% and some of 0.1% were lethal. The seedlings showed necrosis with subsequent death even before new flush emerged (Fig. 2). Some of the surviving seedlings from concentrations of 0.2% and 0.3% showed mixed hyperploidy. Such plants were unstable and ceased to grow after a brief period of time. One of the tetraploids obtained from selection UKP1, and a mixoploid from reverted back to diploid after 8 weeks. The reversions of the tetraploids into diploids showed that they were not stable over time. Colchicine interferes with cell division and the affected cells divide at a slower rate than unaffected cells. Diploid cells generally divide more vigorously than autotetraploid cells, and are probably responsible for most of the seedling growth, and this is the most probable cause of reversion to the diploid form. The stable tetraploid plants confirmed by flow cytometry were micrografted onto vigorous rootstocks for further growth and were moved to the greenhouse (Fig. 2c). Conclusion The tetraploids from elite monoembryonic pummelo selections selected for their red flesh and superior quality may be of significant value in triploid grapefruit/pummelo breeding programs. In this study, a method to induce tetraploidy in pummelo seedlings by treating pre-germinated seeds with colchicine at various concentrations and exposure periods is described. Stable tetraploids were successfully produced from all three selections and were confirmed by flow cytometry. This method facilitates treatment of large number of seeds at the same time perhaps reducing safety risks when working with colchicine since with shoot tip grafting far more handling is necessary as individual shoot tips that have to be treated separately. These tetraploids are potential female parents in interploidal crosses for triploid breeding and will be used to produce red fleshed seedless pummelo/grapefruit types. Literature Cited Barret, H.C Colchicine-induced polyploidy in Citrus. Bot. Gaz. 135: Blakeslee, F.A. and A.G. Avery Methods of inducing doubling of chromosome in plants. J. Hered. 25: Bouvier, L., F.R. Pillon, and Y. Lespinasse Oryzalin as an efficient agent for chromosome doubling of haploid apple shoots in vitro. Plant Breeding 113: Cameron, J.W. and R.H. Burnett Use of sexual tetraploid seed parents for production of tetraploid citrus hybrids. HortScience 13: Chen, R.Z. and L.Y. 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